This proposal is a revised application in response to an RFA for R21 applications to advance Novel Science in Women's Health Research. Although heart disease is the #1 killer of both Men and Women in the US, only 27% of participants in cardiac clinical trials are women. In basic research, most experiments are performed on male animals only. It is therefore not surprising that little is known about the basic molecular mechanisms even for such clinically important fundamental factors such as why female cardiac action potentials are longer than male action potentials. Even less is known about why 70% of congenital Long QT syndrome patients are women, and why women are at particular risk for drug induced arrhythmias. This application proposes to advance science in women's health research by determining the molecular basis for sex dependence and hormonal regulation of IKr and IKs, the two major repolarizing currents in heart. This research is strongly hypothesis driven, and the overall guiding hypothesis is that differences in the electrophysiological and pharmacological profile of IKr and IKs are responsible for sex differences in cardiac repolarization. This is a departure from the current concept that it is purely the action potential duration that is the key determinant of arrhythmia susceptibility. We are proposing that the important factor is not the absolute action potential length, but the relative contribution of IKr and IKs, combined with their pharmacological sensitivities that are a critical factor in arrhythmogenesis. The alpha subunits of IKr and IKs are HERG and KCNQ1 respectively. However, their relative expression, electrophysiological and pharmacological profiles are determined by the presence of KCNE ancillary subunits. Our hypothesis is that hormonal regulation of these subunits is the major factor in determining differences between male and female myocytes. We will test this hypothesis by determining IKr and IKs electrophysiological profile in human cardiomyocytes derived from male and female induced Pluripotent Stem Cells (iPSCs) from skin cells, as well as ventricular myocytes from male, female and ovariectomized (OVX) guinea pigs. We will also expose myocytes to estradiol and testosterone to test the direct effects of hormones on currents and use molecular interventions to determine subunit specificity. We will use the experimental data to develop mathematical models of human and guinea-pig action potentials which contain hormonal regulation of IKr and IKs. We will use these models to make predictions about the dynamic behavior of repolarization (e.g., restitution, QT prolongation and pharmacological sensitivity) which can be tested in our experimental human and guinea pig models. These innovative simulation studies will provide testable hypotheses which are readily applicable to electrophysiological studies in humans. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand the basis of sex-differences in the electrical activity and pharmacological sensitivity of the heart. Sex is an often overlooked fundamental clinical variable, which can have a significant impact on health outcome. Understanding the basis of cardiac sex differences offers the opportunity for translational advances in the identification of therapeutic targets with the potential to improve patient outcomes and improve healthcare for both men and women.